Electrochemical immunoassays using colloidal metal markers

ABSTRACT

The invention concerns a method for detecting or quantifying a biological substance coupled with a colloidal metal particle by electrochemical detection, characterised in that it comprises a step which consists in dissolving by chemical treatment of said colloidal metal particle, prior to detection.

[0001] The detection and quantification of biological substances usingimmunoassay methods or methods for assaying DNA fragments by nucleicacid hybridization are extremely important in many fields of clinicalbiology (medical and biological research, diagnosis, genetics, screeningfor illicit substances in trace amounts, etc.) or even in theenvironmental field (detection of contaminants such as pesticides orbacteria). These methods make it possible to satisfy the double criteriaof selectivity and sensitivity. Among these methods, immunoanalysis witha marker, based on antigen/antibody affinity recognition, isparticularly effective and has become widespread due to the developmentof a large range of nonradioactive markers such as enzymatic,fluorescent or chemiluminescent markers, generally coupled tospectroscopic detection. However, each marker has its own advantages anddisadvantages. Specifically, an ideal marker should satisfy variousrequirements; it should:

[0002] 1) be detectable in a sensitive manner using analyticalinstruments which are inexpensive and easy to manipulate,

[0003] 2) allow the labeled molecule (tracer) to remain soluble andstable in the assaying media,

[0004] 3) allow simple and effective labeling at a reasonable cost,

[0005] 4) having a long lifetime,

[0006] 5) be of no risk to the individual handling it,

[0007] 6) produce a tracer having a reactivity close to the unlabeledmolecule,

[0008] 7) produce a minimal background noise.

[0009] Among the markers which have been marketed, fluorescent andluminescent markers, developed at the beginning of the 1970s, have manyadvantages: they are generally nontoxic and stable, and detectionthereof is very sensitive. However, they require relativelysophisticated and expensive equipment, and the measurement is oftenaffected by endogenous fluorescence associated with sample matrixeffects.

[0010] Enzymatic markers, which appeared at the same time as fluorescentmarkers, are probably today the most popular due to their notablecatalytic properties, but also because of their ability to trigger, forcertain substrates, colored reactions which permit the use of a verysimple detector, such as a calorimeter, or even a manipulator's eye.Enzymatic markers are the basis for methods called ELISA (Enzyme-LinkedImmunoSorbant Assay). These too have their own disadvantages however.Certain substances present in the sample may inhibit the enzyme. Inaddition, they are relatively fragile and have a limited lifetime.Moreover, the background noise can be considerable.

[0011] Metal-based markers were introduced toward the end of the 1970s,with the aim, partly, of remedying certain of the abovementioneddisadvantages. Metal-based markers are distinguished according to theirchemical nature, namely colloidal metal particles, metal ions,coordination complexes, organometallics or else metalloproteins.Depending on their nature, various analytical techniques can beassociated with them, such as time-resolved fluorescence, atomicabsorption spectrophotometry or Fourier transform infrared, or elseelectrochemical techniques such as polarography or voltammetry.

[0012] Compared to spectrophotometric methods, electrochemicaltechniques have many advantages: the measurements can be made in verysmall volumes of liquid (less than a microliter), in medium which ispossibly turbid (in the case of sera), with the possibility of offeringa good sensitivity for inexpensive, possibly portable (small in size)equipment. Although electrochemical techniques make it possible todetect organometallic markers or ionic metals down to nanomolar (10⁻⁹ M)concentrations, this often remains insufficient, however, compared tofluorescent markers which themselves can be detected down to picomolar(10⁻¹² M) thresholds. The electrochemical detection strategy developedin the present invention shows that it is possible to attainconcentrations of a metallic marker of the order of 10⁻¹² M.

[0013] The invention relates more precisely to a method forelectrochemical detection of a colloidal metal particle used as a markerin an immunoassay. The invention also relates to the quantitative orqualitative determination of compounds which may be haptens, antigens orantibodies, but also compounds such as DNA or RNA fragments. In general,the invention may be extended to all analytical methods involving aspecific, affinity interaction between a ligand and a host molecule, andin which it is necessary to add a marker for substantially quantifyingsaid interaction. Furthermore, many formats of immunoassays, whethercompetitive or noncompetitive, or of methods of nucleic acidhybridization, preferably in heterogeneous phase, can be applied.

[0014] The use of colloidal metal particles as a marker is not new.Specifically, they are used very commonly as a contrasting agent inelectromicroscopy techniques, in particular in the form of gold colloidscoupled to antibodies for the purpose, for example, of determining thedistribution of an antigen at the surface of a cell (Beesley,Proceedings RMS, 1985, 20, 187-196). On the other hand, the use of acolloidal metal particle as a marker in the context of an affinity assayis relatively uncommon. In this respect, the existence of a patent (U.S.Pat. No. 4,313,734) and of a publication (Leuvering et al., J.Immunoassay, 1980, 1, 71-91) relating the use of a marker based oncolloidal gold or colloidal silver in the context of an immunoassay withdetection by atomic absorption or colorimetry may be noted. Other quitesimilar patents also relate the use of gold colloid as markers inimmunoassays with calorimetric detection (U.S. Pat. No. 4,853,335,EP0310872, EP0258963). A detection, also calorimetric, has recently beendescribed for the analysis of DNA fragments by hybridization, via acolloidal gold marker (Storhoff et al., J. Am. Chem. Soc. 1998, 120,1959-1964).

[0015] As regards electrochemical detection, a method forimmunoanalyzing or assaying DNA by hybridization, involving theelectrochemical detection or quantification of a colloidal metal marker,does not, for the moment, appear to have been described. The existenceof an article concerning the direct detection of a gold colloid coveredwith antibodies and adsorbed to the surface of a carbon paste electrodecan, however, be reported (Gonzalez-Garcia and Costa-Garcia,Bioelectrochem. Bioenerg. 1995, 38, 389-395). However, application to animmunoassay, although envisioned, was not demonstrated.

[0016] In order to test this hypothesis, the inventors sought to verifywhether or not it was possible to carry out an immunoassay as envisionedby the authors, i.e. an immunoassay taking place at the very surface ofthe electrode and for which, after immunoreaction, the colloidal goldmarker which has reacted in the proximity of the surface of theelectrode is directly detected. The result of this experiment made itpossible to conclude that it was not possible to detect the gold colloidin this way, probably because the latter is no longer in immediatecontact with the surface of the electrode. The present invention makesit possible to be free of this problem by virtue of an indirectdetection of the colloidal metal marker, which by the same token allowsthe use of a solid phase which may be different from the surface of theelectrode.

[0017] Thus, the present invention allows the detection orquantification of a biological substance coupled to a colloidal metalparticle, by electrochemical detection, said colloidal metal particlebeing dissolved, and detected by electrochemistry after having beenreprecipitated at the surface of the electrode. This makes it possibleto increase the local concentration and the sensitivity threshold. Thesubject of the invention is therefore a method for detection orquantification of a biological substance coupled to a colloidal metalparticle, by electrochemical detection, characterized in that itcomprises a step of dissolving said colloidal metal particle.

[0018] The electrochemical immunoassay method developed in the presentinvention can not only be more sensitive than the current enzymaticimmunoassay techniques, but also offer the possibility of determiningand/or quantifying several compounds simultaneously if several colloidalmetal markers which are different in nature are used. Specifically,electrochemical methods permit the simultaneous detection of severalmetals in the course of the same measurement. In addition, colloidalmetal markers offer the advantage of being much more stable thanradioisotopic or enzymatic markers, and they permit simple labeling ofmany substances, at low cost, without any loss of activity of thesesubstances.

[0019] The chemical treatment to dissolve the colloidal metal particleis carried out in an acidic medium containing an oxidant. Theconcentration of oxidant is chosen so as to be in sufficient excess todissolve the highest concentrations of colloidal metal marker. Asolution of hydrobromic acid containing bromine (Br₂) or hypobromousacid (HBrO) or a mixture of the two as oxidant is preferred (forexample: 10⁻⁴ M of Br₂ in 0.1 or 1 M HBr), in particular whendissolution of a gold colloid is desired. A solution of hydrochloricacid (for example 0.1 M) containing a bromide salt (concentration ≧0.1M) and bromine may also be suitable. Depending on the nature of themetal colloid to be dissolved, other acidic media for dissolution(H₂SO₄, HClO₄, HF, etc.) and oxidizing reagents (I₂, Cl₂, HClO, HIO,H₂O₂, HNO₃, CN⁻, Cr₂O₄ ⁻, MnO₄ ⁻, . . . ) may be envisioned.

[0020] After dissolution, an additional treatment may be necessary toremove the excess oxidizing reagent such as bromine. To do this, anexcess of phenol, aniline, hydrazine, oxine or one of their derivatives,or else preferably an excess of 3-phenoxyacetic acid, may be added tothe medium. The latter is preferable since it is less toxic. Aconcentration of 5×10⁻⁴ M is generally sufficient. The bromine may alsobe removed by degassing.

[0021] It may be advantageous to add in solution a reagent capable ofcomplexing the metal ion in order to promote detection thereof. Indeed,complexation can transform a nonelectroactive metal ion into adetectable electroactive compound. In addition, the complexed metal ion,because it has a more marked hydrophobic nature, can adsorb to theelectrode and thus be more detected with more sensitively by adsorptivecathodic stripping voltammetry (van den Berg, Anal. Chim. Acta, 1991,250, 265-276).

[0022] After the metal has dissolved, it is reduced at the surface ofthe electrode, preferably by applying a very negative potential. Thepotential is then varied in order to reoxidize the metal, which thengoes into solution. The intensity of the voltammetric peak (surface)reflects the amount of metal deposited on the electrode, and thereforethe amount of colloidal particles initially present in the solution.This therefore makes it possible to perform the assaying. When particlesconsisting of different metals are used, simultaneous detection ispossible due to the distinct reoxidation potentials of the variousmetals.

[0023] Thus, it is possible to deduce the presence and/or the amount ofbiological substance initially coupled to the colloidal particle, as afunction of the presence and/or amount of metal electrodeposited at thesurface of the electrode.

[0024] The colloidal metal particles may consist of metal, such as gold,silver, copper, platinum, rhodium, palladium, iridium, nickel or ironcolloids, or else of metal compounds, such as, for example, metal oxidesor halides or chalcogenides, such as Ag₂O, AgI, Bi₂O₅, Cd₃P₂, CdS, CdSe,CdTe, Co₂O₃, CrO₃, Cu₂S, HgI₂, MnO₂, PbS, PbO₂, SnO₂, TiO₂, RuO₂, ZnO,ZnS or ZnO₂, or metal hydroxides. In general, any metal or metalcompound which can be detected electrochemically can be envisioned,preferably transition metals (van den Berg, Anal. Chim. Acta, 1991, 250,265-276). Practical requirements mean that preference is given to theuse of metals or metal compounds which are only barely present, or notat all, in the assaying medium, and more particularly those which offerthe best detection limits with respect to the electrochemical techniquesused. The invention has been demonstrated in particular for a goldcolloid.

[0025] The metal-based colloidal particles can be obtained using one ofthe many methods described in the scientific literature (Hayat, Ed,Colloidal gold: principles, methods, and applications; Academic Press:San Diego, Calif., 1991—Mackay and Texter, Eds, Electrochemistry incolloids and dispersions, VCH Publishers: New York, 1992—Murray et al.,J. Am. Chem. Soc. 1993, 115, 8706-8715—Frens, Nat. Phys. Sci. 1973, 241,20-22—U.S. Pat. No. 5,637,508—Weller, Angew. Chem. mnt. Ed. Engl. 1993,32, 41-43—Wang and Herron, J. Phys. Chem. 1991, 95, 525-532). Dependingon the method of production, the particles may be between 1 and 200 nmin size, with a very low dispersion. In the present invention, metalcolloids between 5 and 100 nm are preferred. The use of a particle ofconsiderable size is liable to improve the sensitivity of the assay.

[0026] Depending on the format and the type of assay, the colloidalmetal particle can be coupled to an antibody, a protein receptor, anantigen, a hapten, a protein, a peptide, an oligonucleotide or a nucleicacid fragment (in particular DNA or RNA). The term “coupling” isintended to mean any method of chemical or physical attachment, director indirect, to the surface of the particle, such as a covalent bond oran adsorption via electrostatic interactions, hydrogen bridges, etc.Many coupling protocols have been described (Beesley, Proceedings RMS,1985, 20, 187-196—Oliver, Methods in molecular biology, 1999, 115,331-334). The species then labeled with the metal particle is then usedas a reagent which, in combination with immunochemical reagents based onantibodies, protein receptors, haptens, antigens, proteins, peptides,oligonucleotides, or DNA or RNA fragments, buffer solutions, otherchemical reagents, and an electrode-based electrochemical detectionsystem, will make it possible to assay a given substance.

[0027] The principle of the invention is, by way of example, illustratedin FIG. 1, in the case of a sandwich-type noncompetitive immunoassay(FIG. 1A), and also for a competitive immunoassay (FIG. 1B).

[0028] For the first approach (FIG. 1A), the compound to be determined(the analyte) is initially captured with a first ligand (in the presentcase an antibody; for a hybridization assay, this would be anoligonucleotide) immobilized on a solid phase. The solid phase forimmobilizing the ligand may, for example, be the bottom of amicrocuvette (in the case of FIG. 1), the surface of a microbead(optionally magnetic), the surface of a membrane or else the surface ofthe electrode. After a given incubation period, optionally followed by awashing step, a second ligand (here an antibody) labeled with a metalcolloid is added in such a way that it reacts with the analytepreviously extracted on the solid phase. The solid phase thusconstituted is then washed then treated with an appropriate volume of asolution of reagent able to dissolve the colloidal metal marker whichhas reacted with the solid phase. Then, the metal thus solubilized inthe ionic state is detected and quantified using an electrode eitherimmersed in the solution (in situ method—FIG. 1A), or after transfer ofthe solution (ex situ method). The electrochemical response can then bequalitatively or quantitatively linked to the substance to be assayed.

[0029] In the case of the second approach (FIG. 1B), the method consistsin bringing a sample containing the substance to be assayed into contactwith a known amount of said substance labeled with a metal colloid, anda certain amount of ligand (antibody) immobilized on a solid phase anddirected against this substance. After a given reaction time, the natureand the amount of metal colloid present in the fraction bound is then,after an optional washing step, determined as indicated above afterdissolution and then electrochemical detection.

[0030] The use of a solid phase consisting of microbeads, for examplemade of latex or else of ferromagnetic oxide, may be particularlyadvantageous for improving the sensitivity and lowering the limit ofdetection. Specifically, the microbeads can be concentrated, after theimmunoreaction step, on a small surface, such as, for example, thesurface of a filtering membrane (U.S. Pat. No. 4,853,335—Tu et al.,Anal. Chem. 1993, 65, 3631-3665) or else the bottom of a conical tube,thus offering the possibility of dissolving the metal colloid in asmaller volume of liquid than that in which the immunoreaction tookplace.

[0031] Methods based on agglutination and/or precipitation inhomogeneous phase of the colloidal metal marker in the course of animmunoreaction or oligonucleotide hybridization can also be envisioned(U.S. Pat. No. 5,851,777). In this case, the aggregates formed areisolated and then dissolved and detected as previously.

[0032] As regards the nature of the electrodes, carbon-based electrodesare preferred, and more particularly disk electrodes (FIG. 2A) and stripmicroelectrodes (FIG. 2B) obtained by screen-printing with acarbon-based ink. These electrodes are in fact particularly well suitedsince they can be mass-produced at low cost, and can thereforeoptionally be for single use. In addition, their geometric form and alsotheir size can be readily modifiable. However, other types of electrodemay be used, such as electrodes made of glass carbon, graphite,composite materials containing carbon, carbon fibers, and/or carbonpaste. In addition, the surface of the electrodes can be treatedelectrochemically or chemically in order to improve the sensitivity ofdetection of the dissolved metal (Kalcher, Electroanalysis, 1990, 2,419-433—Kalcher et al., Electroanalysis, 1995, 7, 5-22—Ugo and Moretto,Electroanalysis, 1995, 7, 1105-1113). This may be, by way of example,modification of the surface of the electrode or of the composition ofthe ink, with a polymer capable of attracting metal ions viaelectrostatic interaction or complexation, or else electrochemicalpretreatment of the surface of the electrode. Depositing a mercury filmmay also prove to be advantageous for certain metal ions which aredifficult to detect on a carbon electrode. As regards the method ofproducing the electrodes, the screen-printing technique is preferable,although other methods of industrial production, such as rotogravure,inkjet printing, or optionally photolithography may be adapted.

[0033] According to the characteristics of the invention, the use ofmicroelectrodes, preferably of strip microelectrodes, obtained byscreen-printing is particularly advantageous since they offer thepossibility of carrying out measurements in very small volumes (of theorder of a few microliters) for analytical performances which, in termsof sensitivity and limit of detection of metal ion, are generallyimproved (Wong and Ewing, Anal. Chem. 1990, 62, 2697-2702—Wang et al.,J. Electroanal. Chem., 1993, 361, 77-83—Wang and Armalis,Electroanalysis, 1995, 7, 958-961—Alames-Varela and Costa-Garcia,Electroanalysis, 1997, 9, 1262-1266).

[0034]FIG. 3, which compares the calibration curves (current densities)of AuBr₄ ⁻ in 0.1 M HBr, obtained on a screen-printed stripmicroelectrode with a surface area S=1.7×10⁻⁴ cm² (curve 1) and on ascreen-printed disk macroelectrode with a surface area S=0.0962 cm²(curve 2), confirms better sensitivity in the case of the stripmicroelectrode. These curves were obtained by linear anodic strippingvoltammetry in the following way: (I) electrodeposition of gold at aconstant potential of E=−0.3 V for 300 s, (ii) then linear potentialscan from 0.2 V up to 1.1 V at a rate of 50 mVs⁻¹. The peak current(i_(p)) appears around 1.0 V, linked to oxidation of the gold, and istaken as the analytical response.

[0035] It in fact appears that the use of microelectrodes makes itpossible to obtain deposition of the metal which is more effective thanwith a macroelectrode. Specifically, the use of macroelectrodes requiresthe solution to be agitated in order to ensure that a sufficient amountof metal deposits at the surface of the electrode. Surprisingly, theinventors have observed that the use of microelectrodes makes itpossible to do without this agitation step. This may explain the gain insensitivity observed, although other hypotheses, due to the very natureof the microelectrode (very small in size), may also be envisioned.

[0036] Various techniques of electrochemical analysis may be used toassay the dissolved metal ions. They are preferentially anodic strippingvoltammetry (or polarography) with a potential scan which may be linear,cyclic, square-ware, normal pulse or differential pulse, or with asuperimposed sinusoidal voltage, or else anodic strippingchronopotentiometry. However, other techniques may be used, such as ionexchange voltammetry, adsorptive cathodic stripping voltammetry (orpolarography) with a scan which may be linear, cyclic, square-ware,normal pulse or differential pulse, or with a superimposed sinusoidalvoltage, or else chronoamperometry, chronocoulometry or linear, cyclic,square-wave, normal pulse or differential pulse voltammetry (orpolarography) or voltammetry (or polarography) with a superimposedsinusoidal voltage. These techniques require a possibly two-electrode oreven three-electrode assembly, i.e. an assembly comprising theabovementioned measuring electrode, a reference electrode and,optionally, an auxiliary electrode. In order to avoid contamination ofthe assaying medium with the metal or the electrolyte of the referenceelectrode, it is advantageous to isolate this electrode with anextension which consists, at its end, of a porous material, and which isfilled with an electrolyte. A reference electrode screen-printed usingan ink based on silver and silver chloride may also be envisioned. Hereagain, it may be useful to isolate this electrode via an electrolytebridge, such as, for example, an ionic conducting gel or an ionicconducting polymer, in order to avoid interference from the silver ionsduring a measurement.

[0037] The invention also relates to a kit for assaying at least onebiological compound. According to the characteristics of the invention,this kit comprises at least one reagent labeled with a colloidal metalparticle and at least one electrode. The kit according to the inventionmay also contain at least one reagent for dissolving the colloidal metalparticle and, optionally, a reagent for eliminating the excess oxidizingreagent. The kit may also contain a reagent capable of complexing themetal ion, in order to promote detection thereof. Finally, the kit mayalso contain instructions in order to enable the method according to thepresent invention to be carried out.

[0038] The following examples illustrate the characteristics of theinvention, but should not be considered to limit the invention.

DESCRIPTION OF THE FIGURES

[0039]FIG. 1. Diagrammatic representation of the principle of theinvention illustrated in the case (A) of a noncompetitive immunoassayand (B) a competitive immunoassay.

[0040]FIG. 2. Diagrammatic representation of a screen-printed diskelectrode (A) and microstrip electrode (B).

[0041]FIG. 3. Calibration curves for ionic gold, obtained (1) with astrip microelectrode and (2) with a disk electrode.

[0042]FIG. 4. Diagrammatic representation of the measuring device usedto detect the gold dissolved in a small volume of a drop of solution.

[0043]FIG. 5. Calibration curves for two colloids of gold covered withstreptavidin.

[0044]FIG. 6. (A) log-log calibration curve for the IgG noncompetitiveimmunoassay. (B) anodic stripping voltammetry curves obtained forvarious concentrations of IgG. The curves are identified by letters inorder to make them correspond to the concentrations indicated by thesame letters on the IgG calibration curve.

[0045]FIG. 7. Log-log calibration curve for the α-fetoproteinnoncompetitive immunoassay.

EXAMPLES Example 1

[0046] Detection of Streptavidin Labeled with a Gold Colloid AfterSpecific Attachment to the Bottom of a Microwell

[0047] The experiments are carried out at ambient temperature.

[0048] The bovine serum albumin (BSA, fraction V), thebiotin-amidocaproyl coupled to BSA (B-BSA, biotin content: 8-12 mol/molof BSA), the streptavidin labeled with colloidal gold (S—Au) 20 nm indiameter, and also the streptavidin coupled to albumin onto which 10 nmcolloidal gold particles are adsorbed (SA—Au) come from Sigma ChemicalCo.

[0049] 100 μl of B-BSA at 10 μg ml⁻¹ in a bicarbonate buffer (15 mMNa₂CO₃; pH 9.6) are pipetted at the bottom of a polystyrene microwell(Nunc) an allowed to incubate for 2 hours. After having emptied themicrowell and rinsed it with 110 μl of phosphate buffer (PBS: 4.3 mMNaH₂PO₄, 15.1 mM Na₂HPO₄ and 50 mM NaCl; pH 7.4), 100 μl of PBScontaining 0.1% of BSA (PBS-BSA) are added and allowed to incubate for 2hours. The microwell is then emptied and rinsed 3 times with 110 μl ofpure water. Next, 35 μl of a solution of S—Au or SA—Au at x μg.ml⁻¹(0.003<x<3) in a PBS-BSA buffer containing 0.05% of Tween 20 (PBS-BSA-T)are then introduced into the microwell and allowed to react for 3 hours.Once emptied, the microwell is thoroughly washed with 3×110 μl ofPBS-BSA-T, and then with 2×110 μl of PBS. The gold colloid attached tothe walls of the microwell is then dissolved by introducing 40 μl of asolution of Br₂ at a concentration of 10⁻⁴ M in 1 M HBr. After 5minutes, a volume of 35 μl is removed from the microwell and transferredonto the surface of a screen-printed carbon disk electrode (S=0.0962cm², electrode prepared according to the method described in the ref.:Bagel et al., Anal. Chem. 1997 , 69, 4688-4694), to which are added 5 μlof a fresh solution of 3-phenoxypropionic acid at 4×10⁻³ M in 1 M HBr. Areference electrode (Ag/AgBr, NaBr_(sat)) extended via an extensioncontaining a saturated solution of NaBr, and an auxiliary electrode arethem immersed in the 40 μl of solution previously deposited onto thesurface of the screen-printed carbon electrode, as shown by the diagramin FIG. 4. The linear anodic stripping voltammetry measurements are thencarried out in the following way:

[0050] 1) electrodeposition of the gold at a constant potential ofE=−0.3 V for 300 s,

[0051] 2) then a linear potential scan from 0.2 V up to 1.1 V at a rateof 50 mVs⁻¹.

[0052] The peak current (i_(p)) which appears around 1.0 V, linked tooxidation of the gold, is taken as the analytical response. Themeasurement may also be the integral of the peak, which then correspondsto a coulomb quantity (Q_(p)). The calibration curves are represented inFIG. 5, on a logarithmic scale, for each of the streptavidins labeledwith gold. Better sensitivity with the SA—Au (curve 1) than with theS—Au (curve 2) can be noted.

Example 2

[0053] “Sandwich” Immunoassay for an Immunoglobulin

[0054] The ovalbumin (OA, grade III) and the goat immunoglobulin G (IgG)is marketed by Sigma Chemical Co. The anti-goat IgG labeled with an 18nm gold colloid, and also the unlabeled anti-goat IgG, are polyclonalantibodies from the Jackson Immunoresearch Laboratories.

[0055] 60 μl of a solution of anti-IgG at 24 μg ml⁻¹ in a PBS buffer arepipetted into a microwell and allowed to incubate for 1 hour. Afterhaving emptied the microwell and rinsed it with 2×100 μl of PBS buffercontaining 0.5% of ovalbumin and 0.1% of Tween 20 (PBS-OA-T), 100 μl ofthis same buffer are then added and allowed to incubate for 1 hour. Thesolution is then drawn off, and 35 μl of a solution of goat IgG at xng.ml⁻¹ (0.5<x<1 000) diluted in a PBS buffer containing 0.1% of Tween20 are then introduced and allowed to incubate for 40 minutes. Once themicrowell has been emptied and rinsed with 2×100 μl of PBS-OA-T 100 μlof PBS-OA-T are introduced. After 30 minutes, the liquid is replacedwith 35 μl of a dilute solution of anti-IgG labeled with colloidal gold(45-fold dilution of the solution marketed, in PBS-OA-T), and thenincubated for 3 hours. A final rinsing cycle is carried out, washing themicrowell 3 times with 200 μl of PBS-OA-T, followed by 2×100 μl of PBS.The liquid is then carefully drawn off, and the gold colloid attached tothe walls of the microwell is then dissolved and detected as indicatedin example 1.

[0056] Some examples of measurements obtained by linear anodic strippingvoltammetry are given in FIG. 6A, while the corresponding goat-IgGcalibration curve is represented in FIG. 6B. Each point represents themean of 2 measurements and each measurement was obtained with adifferent electrode (single-use electrode). A concentration ofapproximately 3×10⁻¹² M of IgG could be determined.

Example 3

[0057] Noncompetitive Immunoassay for Human α-fetoprotein

[0058] 80 μl of a solution of monoclonal anti-α-fetoprotein (mouseantibody) at 24 μg ml⁻¹ in a carbonate buffer (15 mM, pH 9.6) arepipetted into a microwell and allowed to incubate overnight at 4° C.After having emptied the microwell and rinsed it with 2×250 μl ofPBS-OA-T buffer, 250 μl of this same buffer are then added and allowedto incubate for 40 min. The solution is then drawn off, and 80 μl of asolution of α-fetoprotein at x ng.ml⁻¹ (0.05<x<20) diluted in a PBSbuffer containing 0.1% of Tween 20 are then introduced and allowed toincubate for 2 hours. Once the microwell has been emptied and rinsedwith 2×250 μl of PBS-OA-T, 250 μl of PBS-OA-T are introduced. After 30minutes, the liquid is replaced with 80 μl of a dilute solution ofpolyclonal antip-α-fetoprotein (goat antibody) diluted to 5 μl ml⁻¹ inPBS-OA-T, and incubated for 1 hour. Then, after rinsing with 2×250 μl ofPBS-OA-T buffer, 50 μl of a solution of anti-goat IgG labeled withcolloidal gold (45-fold dilution of the solution marketed, in PBS-OA-T)[lacuna], and then incubated for 1 h 30 min. A final rinsing cycle iscarried out, washing the microwell 3 times with 250 μl of PBS-OA-T,followed by 2×250 μl of PBS-T, then 2×250 μl of PBS. The liquid is thencarefully drawn off, then the gold colloid attached to the walls of themicrowell is then dissolved and detected with strip microelectrodes inthe following way: 50 μl of a solution of 0.1 mM Br₂ in 0.1 M HBr areadded to the microwells for 30 min; then 40 μl are transferred into newmicrowells containing 10 μl of a solution of 3-phenoxypropionic acid at2×10⁻³ M in 0.1 N HBr. Detection of the dissolved gold is then carriedout as indicated in example 1.

[0059] Production of Screen-Printed Strip Microelectrodes:

[0060] The carbon-based ink used to produce the strip microelectrodes isa commercial ink produced by Acheson Colloid (Minico® inks of theM3000-1RS series or Electrodag® inks such as 423 SS or PF 407A). The inkis screen-printed onto a rigid or semi-rigid support, preferably made ofsemicrystalline polystyrene or high-impact polystyrene (plates possiblybetween 0.1 and 2 mm thick). The fineness of the screen-printing maskobtained on a taut screen mounted on a frame, and also the nature andthe mesh size of the screen, condition to a large extent the quality ofthe ink deposit and also its thickness. In the present invention, inkthicknesses of between 5 and 50 μm could be obtained usingscreen-printing frames comprising 77 or 120 threads/cm.

[0061] Once the ink has been screen-printed, it is left to dry in anoven at between 60 and 100° C. Next, a polystyrene-based isolating layeris deposited or screen-printed in such a way as to re-cover part of thecarbon ink previously screen-printed (FIG. 2). After drying, theelectrode thus constituted is cut up transversely along its thickness soas to reveal on the section a strip of carbon of micrometric thickness(depending on the thickness of carbon ink initially screen-printed) andof millimetric length (depending on the length of the motif of theelectrode initially selected) (FIG. 2B).

1. A method for detection or quantification of a biological substancecoupled to a colloidal metal particle, by electrochemical detection,characterized in that it comprises a step of dissolving said colloidalmetal particle, by chemical treatment, before detection.
 2. The methodas claimed in claim 1, characterized in that the dissolving of thecolloidal metal particle is carried out in an acidic medium containingan oxidant.
 3. The method as claimed in either of claims 1 and 2,characterized in that the step of dissolving the colloidal metalparticle is followed by an additional treatment intended to eliminatethe product inducing said dissolution.
 4. The method as claimed in oneof claims 1 to 3, characterized in that a reagent capable of complexingthe metal ion is added in solution.
 5. The method as claimed in one ofclaims 1 to 4, characterized in that the step of dissolving thecolloidal metal particle is followed by a step of reduction and/orprecipitation of the metal at the surface of an electrode.
 6. The methodas claimed in claim 5, characterized in that the reduction and/orprecipitation of the metal on the electrode is carried out using asuitable negative potential.
 7. The method as claimed in either ofclaims 5 and 6, characterized in that the amount of metal precipitatedat the surface of the electrode is measured by variation of thepotential of said electrode and analysis of the voltammetric peak whichappears after reoxidation of said metal and redissolving thereof.
 8. Themethod as claimed in one of claims 1 to 7, characterized in that thecolloidal particle is chosen from particles consisting of metal or ofmetal compounds.
 9. The method as claimed in claim 8, characterized inthat the colloidal particle consists of gold.
 10. The method as claimedin one of claims 1 to 9, characterized in that the colloidal particle isbetween 1 and 200 nm in size.
 11. The method as claimed in one of claims2 to 10, characterized in that the acidic medium containing an oxidantis a solution of hydrobromic acid containing bromine, hypobromous acidor a mixture of these two compounds.
 12. The method as claimed in one ofclaims 1 to 11, characterized in that the electrode used is ascreen-printed electrode, in particular a strip microelectrode.
 13. Themethod as claimed in one of claims 1 to 12, characterized in that thesurface of the electrode is treated electrochemically or chemically inorder to improve the sensitivity of detection of the dissolved metal.14. The method as claimed in one of claims 1 to 13, characterized inthat the colloidal particles present in solution after the biologicalreaction are concentrated before dissolution.
 15. The method as claimedin one of claims 1 to 14, characterized in that the biological substancecoupled to the colloidal particle is included in the group consisting ofantibodies, protein receptors, antigens, haptens, proteins, peptides,oligonucleotides, and nucleic acid fragments.
 16. A diagnostic kit,characterized in that it comprises at least one reagent labeled with acolloidal metal particle and at least one electrode, and also an agentfor chemically dissolving said colloidal metal particle.